Members of the ligand-induced nuclear receptor superfamily,including steroid receptors are medically important regulators ofcellular activity. Hormone activated nuclear receptors regulatetranscription of their cognate target genes by binding tospecific DNA recognition sites. Recently, receptors were shown tobe highly regulated in their subcellular distribution. Presumably, the ligand-induced activation process includesreceptor redistribution between cytoplasm and nucleus and withinthe nucleus. The development of chimeras between fluorescentproteins and the nuclear receptors provides a new approach toaddress the roles of receptor trafficking in hormone actions. In collaboration with Dr. Hagers laboratory (NCI) we exploredglucocorticoid receptor functions with a green fluorescentprotein chimera of the rat glucocorticoid receptor (rGFP- GR).Transient expression of rGFP-GR in mouse adenocarcinoma cellsallowed us to study receptor motion and intranuclear targeting.We demonstrated that unliganded GR resides in the cytoplasm andthat hormone addition initiates a cytoplasm to nucleustranslocation. We detected distinct subnuclear localizations ofagonist (dexamethasone) and antagonist (RU486) activatedreceptors. After the addition of dexamethasone, rGFP-GRaccumulated in a discreteseries of foci, while after addition of RU486, rGFP-GR did notform brightfoci but remained in a reticular pattern. We continued to userGFP-GR plasmid and also began usinghuman GFP-GRalpha and human GFP-GRbeta in collaboration with Dr.Chrousoss laboratory (NICHD) to study receptor translocation andintranuclear targeting. Direct observation of GFP-GR binding tobiologically relevant regulatory sites in chromatin wasvisualized in an adenocarcinoma cell line (904.13; gift from Dr.Hager). These cellscarry a minichromosome, a highly amplified set of mouse mammarytumor virus reporter elements that contain 800 GR binding sitesin a head-to-tail tandem repeat. We established sublones thatstably express hGFP-GRalpha in this 904.13 cells. Agonistexposure caused both rat and human GFP-GR to accumulate along theMMTV-LTR array, appearing as a bright object having a 100-foldlarger volume than other hormone-induced foci. We also calculatedGFP-GR motion rates within the nucleus and residence time ontarget genes with fluorescence recovery after photobleaching.These studies showed that ligand-activated GR bind totarget sites for less than a minute. We generatedtranscriptionally inactive DNA-binding zinc-finger mutants of rGFP-GR. These mutant receptors translocated into the nucleusafter dexamethasone addition, but remained in fast motion withinthe nucleus and failed to bind to the array. Ligand-activatedGFP-VDR and EYFP-RXR did not bind to the array, while they alsoformed bright foci. The intranuclear motion rate of VDR wassimilar to the rate of GR, and the residence time of VDR on fociwas less than 3 min. DNA-binding mutants of VDR remained in fastmotion and failed to form foci. These experiments revealedsimilarities in the intranuclear targeting mechanisms for nuclearreceptors.We will continue to study the subcellular and subnucleardistribution of the receptor family members to defineprotein-protein interactions that regulate receptor motion and toaddress important questions regarding selective hormone actions. - Steroid receptors, chromatin, green fluorescent protein, protein trafficking, translocation, glucocorticoid receptor, MMTV, hormone actions